反应详情

Keyhole limpet hemocyanin (KLH, 60 mg) was reconstituted in 100 mM sodium phosphate buffer at pH 7.2. 2-Iminothiolane (2IT, 13.5 mg) was added to the protein solution as a solid, and the reaction was allowed to stir at room temperature in the dark and under argon atmosphere for 1 hour. The activated KLH-(SH)n was desalted on a Sephadex PD-10 column pre-equilibrated with 100 mM sodium phosphate buffer at pH 6.5. The SH loading was determined (Ellman's reagent) to be 886 per KLH molecule (MW 5,000,000). To 6 ml of KLH-(SH)n, 4.7 mg/mL, was added a solution of 14 mg (0.027 mmol) of MDEA-maleimide (2O) in 1 mL of DMF dropwise, and the mixture was allowed to stir at room temperature 18 hours. The resulting conjugate was placed in a dialysis tube (10,000 MW cut-off) and was dialyzed in 1 L of phosphate buffered saline (PBS) buffer (180 mM NaCl, 10 mM sodium phosphate, pH 7.2] containing 20% DMF. (3 times, at least 6 hours each). This was followed by 1 L of PBS buffer, pH 7.2 at 4° C. The protein concentration was determined to be 2.08 mg/mL using Biorad Coomassie blue protein assay (Bradford, M., Anal. Biochem. 72:248, 1976). A total of 20 mL of the conjugate was obtained.

1. stirringto stir at room temperature 18 hours
2. customThe resulting conjugate was placed in a dialysis tube
3. custom(10,000 MW cut-off) and was dialyzed in 1 L of phosphate buffered saline (PBS) buffer (180 mM NaCl, 10 mM sodium phosphate, pH 7.2]
4. additioncontaining 20% DMF
5. custom3 times, at least 6 hours
6. customThis was followed by 1 L of PBS buffer, pH 7.2 at 4° C
7. concentrationThe protein concentration
8. customA total of 20 mL of the conjugate was obtained